The long term goal of this project is to understand the molecular and cellular mechanisms by which cytotoxic lymphocytes (CL) kill their target cells in health (eg. viral and tumor clearance) and disease (eg. Graft vs. Host Disease, and autoimmune states). CL can kill by secreting the contents of their cytotoxic granules (including perforin and granzymes) onto the surface of target cells via the "secretory synapse." Perforin is responsible for delivering and/or trafficking the granzymes, which induce target cell death by cleaving a variety of substrates. Granzymes A and B induce cell death via non-overlapping pathways, but several "orphan" granzymes are expressed in both mice and humans, and may be relevant for specific CL functions. We have made (or are currently making) pure 129/SvJ mice deficient for granzymes A, B, C (and all combinations thereof), and perforin. With this unique set of reagents, we will further examine granzyme activities and substrates via the following specific aims: Specific Aim 1: We will define the roles of individual granzymes for perforin-mediated cytotoxity in vivo. Perforin is required for the clearance of many tumors and viruses, and for CDS8+ mediated GvHD, but the roles of individual granzymes in these processes remains extremely controversial. We will use the precisely strain-matched mice described above to better define the roles of these granzymes for CD8+ and NK mediated killing in vitro and in vivo. Specific Aim 2: We will define the role of the perforin/qranzyme pathway for CD4+ mediated immune regulation in mice. We have shown that human regulatory T cells contain perforin and granzymes, and that they can kill autologous immune targets in a perforin-dependent fashion. We will use precisely strainmatched 129/SvJ mice deficient for perforin or granzymes to assess the importance of these molecules for T regulatory cell function in vitro and in vivo. Specific Aim 3: We will use proteomic approaches to define the substrates of qranzymes. We will use 2-D differential in-gel electrophoresis (2D-DIGE) and mass spectrometry to identify the substrates and cleavage products of granzymes A, B, and C. Novel substrates will be evaluated for their importance in mediating granzyme-induced death in vitro and in vivo.